Enzymatic Mechanism of Oxalate Decarboxylase Revealed by Biophysical and Structural Studies

生物物理和结构研究揭示草酸脱羧酶的酶机制

• 批准号: 2002950
• 负责人: Alexander Angerhofer
• 金额: $ 47.3万
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2002950&HistoricalAwards=false
• 关键词: Enzymatic; Mechanism; Oxalate; Decarboxylase; Revealed

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Alexander Angerhofer from the University of Florida to investigate the degradation of oxalic acid by an enzyme known as oxalate decarboxylase which is found in soil bacteria and fungi. Overloading the body with oxalic acid can lead to kidney stones and other adverse health effects in animals and humans. As a plant product oxalic acid is present in many plant-derived foods in appreciable quantities. A way to keep the amount of oxalic acid low is to decompose it by chemical reactions catalyzed by enzymes. The research of Dr. Angerhofer is focused on the elucidation of the mechanism by which the enzyme oxalate decarboxylase breaks down oxalic acid. More specifically, Dr. Angerhofer will test the hypothesis that to decompose oxalic acid, this protein transfers electrons over a long distance, about 2 nm, between two manganese ions, one of which is located at the active site for the chemical transformation of oxalic acid. This study will involve use of a combination of advanced biochemical methods of protein engineering with structural and spectroscopic methods, including X-ray crystallography, electron paramagnetic resonance, and optical spectroscopy. The results of this research will enhance our fundamental knowledge of reactions that involve bio-active manganese and will ultimately aid in devising strategies to mitigate the presence of oxalic acid in plants. The project supports the training of a growing and diverse science and technology workforce in the state of Florida by involving students in research that helps them acquire scientific and professional skills useful in the bio-economy of the 21st century. The training of undergraduate students is supported through a course-based undergraduate research experience at the University of Florida and through the University Research Scholars Program, which brings gifted freshmen students to the cutting edge of modern research.This award supports the research of Dr. Angerhofer focused on the study of the molecular mechanisms by which the bacterial enzyme oxalate decarboxylase catalyzes the cleavage of the kinetically inert carbon-carbon bond in oxalic acid. It has been proposed that long-range electron transfer between the manganese ions situated at the N- and the C-terminal ends of the proteins subunits plays an important catalytic role. X-ray crystallography, molecular dynamics simulations, electrochemistry, and an array of advanced electron paramagnetic resonance (EPR) technologies, in combination with site-directed mutagenesis will be applied to the problem. The activity of manganese ions in the protein and a chain of electron transfer-active amino acids will be studied. Genetic code expansion methods will be used to introduce unnatural amino acids into the enzyme at specific target sites where they can be used to probe a long-range electron transfer pathway that may exist in the quaternary structure of the enzyme. The planned experiments will yield important structural and kinetic information about substrate and oxygen co-factor binding to the enzyme. The hypothesis that will be tested is that via long-range electron transfer dioxygen promotes catalysis by generating the +3 oxidation state on the active-site Mn ion situated at the N-terminal end, which in turn drives the reaction. The proposed work will elucidate how proteins tune and utilize the redox potential of mono-nuclear Mn centers for catalysis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

有了这个奖项，化学过程的化学过程计划是为佛罗里达大学的Alexander Angerhofer博士提供资金，以研究一种被称为草酸脱羧酶的酶降解草酸，该酶在土壤细菌和真菌中发现。用草酸超载人体会导致肾结石和其他不良健康影响。由于植物产物的草酸存在于许多植物来源的食物中，数量可观。保持草酸含量低的一种方法是通过酶催化的化学反应将其分解。 Angerhofer博士的研究重点是阐明草酸酶脱羧酶分解草酸的机制。更具体地说，Angerhofer博士将检验以下假设：要分解草酸，该蛋白质将电子在两个锰离子之间转移约2 nm，其中一个位于活性位点，用于用于草酸化学转化。 这项研究将涉及将蛋白质工程的先进生化方法与结构和光谱方法的结合组合，包括X射线晶体学，电子顺磁共振和光谱法。这项研究的结果将增强我们对涉及生物活化锰的反应的基本知识，并最终将有助于制定策略来减轻植物中草酸的存在。该项目支持培训佛罗里达州不断增长和多样化的科学和技术劳动力，通过参与研究，这有助于他们获得对21世纪生物经济有用的科学和专业技能。通过在佛罗里达大学和大学研究学者计划的基于课程的本科研究经验的支持下，对本科生的培训得到了支持，该计划将有才华的学生带到现代研究的最前沿。草酸中的碳碳键。已经提出，位于蛋白质亚基N-和C末端的锰离子之间的远距离电子转移起着重要的催化作用。 X射线晶体学，分子动力学模拟，电化学和一系列晚期电子顺磁共振（EPR）技术，将与位置定向的诱变结合使用。将研究锰离子在蛋白质和电子转移活性氨基酸链中的活性。遗传代码扩展方法将用于将非天然氨基酸引入特定目标位点的酶中，在该酶中，它们可用于探测酶的Quaternary结构中可能存在的远距离电子转移途径。计划的实验将产生有关底物和氧气因子与酶结合的重要结构和动力学信息。 将要检验的假设是，通过远程电子转移二恶英，通过在位于N末端的活性位点Mn离子上产生+3氧化态，从而促进催化，这反过来驱动了反应。拟议的工作将阐明蛋白质如何调整和利用单核MN中心的氧化还原潜力进行催化。该奖项反映了NSF的法定任务，并被认为是通过基金会的智力优点和更广泛影响的审查标准通过评估来获得支持的。

项目成果

Selective incorporation of 5‐hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

选择性掺入 5-羟色氨酸可阻断草酸脱羧酶中的长程电子转移

• DOI: 10.1002/pro.4537
• 发表时间: 2022
• 期刊: Protein Science
• 影响因子: 8
• 作者: Pastore, Anthony John;Montoya, Alvaro;Kamat, Manasi;Basso, Kari B.;Italia, James S.;Chatterjee, Abhishek;Drosou, Maria;Pantazis, Dimitrios A.;Angerhofer, Alexander
• 通讯作者: Angerhofer, Alexander

Epoxyqueuosine Reductase QueH in the Biosynthetic Pathway to tRNA Queuosine Is a Unique Metalloenzyme.

• DOI: 10.1021/acs.biochem.1c00164
• 发表时间: 2021-10-26
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Li Q;Zallot R;MacTavish BS;Montoya A;Payan DJ;Hu Y;Gerlt JA;Angerhofer A;de Crécy-Lagard V;Bruner SD
• 通讯作者: Bruner SD